Biomedical subjects

Bryan D Young

Publications and source records attributed to Bryan D Young.

34 records · Page 2Linked to original sources

PubMed2004

s-thalidomide has a greater effect on apoptosis than angiogenesis in a multiple myeloma cell line.

s-Thalidomide has proven efficacy in multiple myeloma. Although it has both antiangiogenic and pro-apoptotic effects, its primary mode of therapeutic action remains unclear. We have investigated the changes to the expression of genes involved with these cellular processes following culture with s-thalidomide in the U266 MM cell line. Cells were cultured with s-thalidomide (0-1000 microM), and cell parameters, including apoptosis, were assessed on day 3. RNA was extracted from cells cultured for 24 h at the IC(50) concentration of s-thalidomide, and changes to gene expression were investigated by microarray methodologies. A reduction in cell viability was observed in U266 cells cultured with s-thalidomide (IC(50): 362 microM), which were mirrored by significant increases in apoptosis (for example, 200 microM on day 3: 40.3+/-3.1% vs. 3.2+/-0.4% on day 0; P<0.001). There were changes in the expression profile of genes involved in angiogenesis and apoptosis, but the changes were most dramatic in the apoptotic genes. In particular, the expression of I-kappaB kinase was decreased by two-fold, which was associated with a four-fold decrease in NF-kappaB expression. These data correlated with immunoblotting analyses, which showed significant increases in I-kappaB protein levels and decreased NF-kappaB activity. Additionally, the Bax : Bcl-2 ratio was significantly increased. Our data suggest that both angiogenic and apoptotic genes and proteins are affected by s-thalidomide. Additionally, a dramatic decrease in Bcl-2 expression with s-thalidomide suggests a possible enhancement of cytotoxic effect if combined with other cytotoxic agents.

Angiogenesis Inhibitors↗

PubMed2003

Replication timing of the human genome.

We have developed a directly quantitative method utilizing genomic clone DNA microarrays to assess the replication timing of sequences during the S phase of the cell cycle. The genomic resolution of the replication timing measurements is limited only by the genomic clone size and density. We demonstrate the power of this approach by constructing a genome-wide map of replication timing in human lymphoblastoid cells using an array with clones spaced at 1 Mb intervals and a high-resolution replication timing map of 22q with an array utilizing overlapping sequencing tile path clones. We show a positive correlation, both genome-wide and at a high resolution, between replication timing and a range of genome parameters including GC content, gene density and transcriptional activity.

Base Composition↗

PubMed2003

Comparative genomic hybridization of 49 primary retinoblastoma tumors identifies chromosomal regions associated with histopathology, progression, and patient outcome.

Forty-nine primary retinoblastoma (Rb) tumors were analyzed by the use of comparative genomic hybridization (CGH), and clinical/histological correlations were performed. Adverse histological factors were present in 13 patients. Chromosomal imbalance was a frequent phenomenon, seen in 96% of the tumors. Gain of 6p represented the most frequent event (69% of the tumors), whereas +1q was observed in 57%, confirming that these abnormalities are key secondary events in retinoblastoma tumor progression. Loss of 13q and 16 was significantly associated with tumors displaying adverse histo-prognostic factors, whereas -16q was significantly associated with tumors without adverse features. In three patients who developed an extra-ocular relapse, the tumors showed -13q and 2/3 had -5q, suggesting that these abnormalities may be associated with metastasis. Children >or= 36 months of age at enucleation tended to have more CGH abnormalities per tumor than children < 12 months (median numbers 11 vs. 3). In addition, +1q, +13q, -16, and -16q were more frequent in children with an older age at enucleation. Identical CGH changes were found in both tumors from one patient with bilateral tumors, suggesting a common origin. It is possible that tumors displaying loss of 13q and 5q indicate those patients who may suffer an adverse outcome and who would require alternative or more intensive therapy. CGH analysis on larger cohorts and in prospective clinical trials will be invaluable in determining whether a genetic classification of retinoblastoma represents a reliable measure of prognosis.

Child↗

PubMed2003

Molecular cytogenetic characterization of Sézary syndrome.

Sézary syndrome (SS) is a rare form of erythrodermic cutaneous T-cell lymphoma with hematological involvement and a poor prognosis. At present little is known about the molecular pathogenesis of this malignancy. To address this issue, we analyzed 28 SS cases through the use of molecular cytogenetic techniques. Conventional cytogenetic analysis showed 12 of 28 cases with clonal chromosome abnormalities (43%). Seven cases had aberrations affecting chromosomes 1 and 17; five demonstrated rearrangement of chromosomes 10 and 14; four presented with an abnormality of 6q. Multiplex-fluorescence in situ hybridization (M-FISH) revealed complex karyotypes in 6 of 17 cases (35%), and recurrent der(1)t(1;10)(p2;q2) and der(14)t(14;15)(q;q?) translocations were each identified in two cases, and confirmed by dual-color FISH. There was an overall difference in the incidence of clonal abnormalities detected by G-banded karyotyping and M-FISH. In addition, comparative genomic hybridization studies revealed chromosome imbalances (CIs) in 9 of 20 cases (45%), with a mean DNA copy number change per sample of 1.95 +/- 2.74, and losses (mean: 1.25 +/- 1.77) more frequent than gains (mean: 0.7 +/- 1.26). The most common CIs noted were loss of 1p, followed by losses of 10/10q, 17p, and 19, and gains of 17q and 18. Furthermore, in conjunction with this study a systematic literature review was conducted, which showed a high frequency and consistent pattern of chromosome changes in SS. These findings suggest that chromosomal instability is common in SS, although there are specific chromosomal abnormalities that appear to be characteristic, and the identification of two different recurrent chromosome translocations provides the basis for further studies.

Chromosome Aberrations↗

PubMed2003

Genetic alterations in primary cutaneous CD30+ anaplastic large cell lymphoma.

Primary cutaneous CD30+ anaplastic large cell lymphoma (C-ALCL) represents a distinct clinical subtype of CD30+ anaplastic large cell lymphomas. The etiology and underlying molecular pathogenesis of C-ALCL remain unclear. This study aimed to investigate genetic changes in C-ALCL. Comparative genomic hybridization (CGH) analysis of 23 DNA samples from 15 C-ALCL cases identified chromosome imbalances (CI) in 10 samples from eight cases (43%). The mean number of CI per sample was 2.09 +/- 3.86, with gains (2.00 +/- 3.85) more common than losses (0.09 +/- 0.29). The most frequent CI were gains of 1/1p and 5 (50%) and 6, 7, 8/8p, and 19 (38%). Microarray-based CGH analysis of six DNA samples from five cases with CI revealed genomic imbalances (GI) in all of the cases studied. This included oncogene copy number gains of FGFR1 (8p11) in three cases, and NRAS (1p13.2), MYCN (2p24.1), RAF1 (3p25), CTSB (8p22), FES (15q26.1), and CBFA2 (21q22.3) in two cases. Real-time PCR analysis of nine DNA samples from eight cases with CI and GI detected amplifications of CTSB and RAF1 in seven cases (88%), REL (2p13p12) and JUNB (19p13.2) in six cases (75%), and MYCN and YES1 (18p11.3) in four cases (50%). Immunohistochemical staining of paraffin sections from six cases demonstrated expression of JUNB protein in five cases and BCL2 in three cases. These results reveal a consistent pattern of genetic alterations in C-ALCL and provide the molecular basis for further investigation of this disease.

Chromosome Aberrations↗

PubMed2003

Genome-wide analysis of acute myeloid leukemia with normal karyotype reveals a unique pattern of homeobox gene expression distinct from those with translocation-mediated fusion events.

Gene expression profiles were determined from presentation peripheral blood and bone marrow samples of 28 patients with acute myeloid leukemia (AML). Hierarchical clustering sorted the profiles into separate groups, each representing one of the major cytogenetic classes in AML [i.e., t(8;21), t(15;17), inv(16), 11q23, and normal karyotype]. Statistical group comparison identified genes whose expression was strongly correlated with these chromosomal classes. Moreover, the normal karyotype AMLs were characterized by distinctive up-regulation of certain members of the class I homeobox A and B gene families, implying a common underlying genetic lesion. These data reveal novel diagnostic and therapeutic targets and demonstrate the potential of microarray-based dissection of AML.

Adult↗

PubMed2003

Molecular cloning of a constitutional t(7;22) translocation associated with risk of hematological malignancy.

We report the molecular characterization of a reciprocal constitutional translocation t(7;22)(p13;q11.2) carried by three family members who have each developed a hematological malignancy. The chromosome 7 breakpoint was localized to a single BAC clone, RP11-571N3, by sequential fluorescence in situ hybridization analysis of clones selected from the NCBI chromosome 7 map. This was further refined to a 739-bp region by Southern blot analysis of DNA from the two cell lines 1193 and 1194 digested with EcoRI, HindIII, PstI, and PvuII. A 2.8-kb fragment spanning the der(22) breakpoint was amplified by long-range inverse PCR. The sequence of this fragment was used to predict the composition of the der(7) breakpoint, and a 1.3-kb fragment was amplified by use of primers from both chromosomes 7 and 22 based on this prediction. The breakpoint on chromosome 22 is located between the 3rd and 4th V regions of the immunoglobulin lambda (IGL) locus, and the breakpoint on chromosome 7 is located 122 kb proximal to the insulin-like growth factor binding protein (IGFBP) 3 gene. Examination of both reciprocal junctions showed that four bases were lost from chromosome 22, whereas 75 bases were lost from chromosome 7. Small insertions of 46 bases and 13 bases were found at the der(22) and the der(7) junctions, respectively. As a consequence of this event, the entire IGL locus, less the first three Vlambda elements, is translocated to chromosome 7, whereas the three remaining Vlambda elements on the der(22) are juxtaposed with IGFBP3 and IGFBP1.

Aged↗

PubMed2003

Genomic alterations in blastic natural killer/extranodal natural killer-like T cell lymphoma with cutaneous involvement.

Natural killer and natural killer-like T cell lymphomas represent a rare type of non-Hodgkin's lymphoma originally described to involve the upper aerodigestive tract. This malignancy has been increasingly observed in other extranodal sites, particularly in the skin. Patients with cutaneous natural killer cell lymphoma generally have a poor prognosis; however, the etiology and the underlying molecular pathogenesis remain unclear. This study aimed to investigate comprehensively genomic changes in blastic natural killer and extranodal natural killer-like T cell lymphoma with cutaneous involvement. Comparative genomic hybridization showed chromosome imbalances in six of eight cases studied (75%). The mean number of chromosome imbalances per sample was 2.18+/-1.63 with similar number of gains (1.18+/-1.17) and losses (1.00+/-1.34). The most frequent DNA copy number changes observed were losses of 9/9p (83%), followed by loss of 13q and gain of 7 (67%). Similar patterns of chromosome imbalances were observed in both blastic natural killer and cutaneous natural killer-like T cell lymphomas. Loss of the RB1 gene at 13q14.2 was detected in one blastic natural killer cell lymphoma with 13q loss using a gene dosage assay, and in one cutaneous natural killer-like T cell lymphoma without 13q loss using fluorescent in situ hybridization. Genomic microarray analysis identified oncogene copy number gains of PAK1 and JUNB in three of four cases studied, and gains of RAF1, CTSB, FGFR1, and BCR in two cases. Real-time polymerase chain reaction detected amplification of CTSB and RAF1 in four of five cases analyzed, JUNB and MYCN in three cases, and REL and YES1 in two cases, respectively. In conjunction with this study, an extensive literature search for the published G-banded karyotypes of four subsets of natural killer cell lymphomas was conducted, which showed a nonrandom pattern of multiple chromosome aberrations. These results reveal consistent genetic alterations in cutaneous natural killer cell lymphomas, and provide a basis for further investigation of molecular pathogenesis in this malignancy.

Chromosome Aberrations↗

PubMed2003

The apparent uptake of fluorescently labeled siRNAs by electroporated cells depends on the fluorochrome.

Transfection of mammalian cells with preformed small interfering RNAs (siRNAs) permits a transient and often specific reduction of gene expression. It is possible to rapidly examine the uptake of siRNAs by transfection with fluorescently labeled siRNAs. We examined the apparent uptake of such siRNAs by several leukemic cell lines after electroporation. We show that Cy3 and Cy5-labeled siRNAs cause a significant amount of cell fluorescence, as judged by flow cytometry. In contrast, several fluorescein-labeled siRNAs could not be detected. Nevertheless, such fluoresceinated siRNAs efficiently suppressed a leukemic target gene, demonstrating that siRNA uptake must have taken place. Therefore, for cell electroporation, fluorescein-labeled siRNAs may lead to false negative results and should not be used to examine electroporation-mediated siRNA uptake.

Base Sequence↗

PubMed2002

Amplification and overexpression of JUNB is associated with primary cutaneous T-cell lymphomas.

Primary cutaneous lymphomas (PCLs) represent a heterogeneous group of extranodal T- and B-cell malignancies. The underlying molecular pathogenesis of this malignancy remains unclear. This study aimed to characterize oncogene abnormalities in PCLs. Using genomic microarray, we detected oncogene copy number gains of RAF1 (3p25), CTSB (8p22), PAK1 (11q13), and JUNB (19p13) in 5 of 7 cases of mycosis fungoides (MF)/Sezary syndrome (SS) (71%), gains of FGFR1 (8p11), PTPN (20q13), and BCR (22q11) in 4 cases (57%), and gains of MYCL1 (1p34), PIK3CA (3q26), HRAS (11p15), MYBL2 (20q13), and ZNF217 (20q13) in 3 cases (43%). Amplification of JUNB was studied in 104 DNA samples from 78 PCL cases using real-time polymerase chain reaction. Twenty-four percent of cases, including 7 of 10 cases of primary cutaneous CD30(+) anaplastic large-cell lymphoma (C-ALCL), 4 of 14 MF, 4 of 22 SS, and 2 of 23 primary cutaneous B-cell lymphoma (PCBCL) showed amplification of JUNB, and high-level amplification of this oncogene was present in 3 C-ALCL and 2 MF cases. JUNB protein expression was analyzed in tissue sections from 69 PCL cases, and 44% of cases, consisting of 21 of 23 SS, 6 of 8 C-ALCL, 5 of 10 MF, and 9 of 21 PCBCL, demonstrated nuclear expression of JUNB by tumor cells. Overexpression of JUNB also was detected in 5 C-ALCL and 2 SS cases. These results have revealed, for the first time, amplification and expression patterns of JUNB in PCL, suggesting that JUNB may be critical in the pathogenesis of primary cutaneous T-cell lymphomas.

DNA, Neoplasm↗

PubMed2002

The AF10 leucine zipper is required for leukemic transformation of myeloid progenitors by MLL-AF10.

The t(10;11)(p12;q23) chromosomal translocation in human acute myeloid leukemia results in the fusion of the MLL and AF10 genes. The latter codes for a novel leucine zipper protein, one of many MLL fusion partners of unknown function. In this report, we demonstrate that retroviral-mediated transduction of an MLL-AF10 complementary DNA into primary murine myeloid progenitors enhanced their clonogenic potential in serial replating assays and led to their efficient immortalization at a primitive stage of myeloid differentiation. Furthermore, MLL-AF10-transduced cells rapidly induced acute myeloid leukemia in syngeneic or severe combined immunodeficiency recipient mice. Structure/function analysis showed that a highly conserved 82-amino acid portion of AF10, comprising 2 adjacent alpha-helical domains, was sufficient for immortalizing activity when fused to MLL. Neither helical domain alone mediated immortalization, and deletion of the 29-amino acid leucine zipper within this region completely abrogated transforming activity. Similarly, the minimal oncogenic domain of AF10 exhibited transcriptional activation properties when fused to the MLL or GAL4 DNA-binding domains, while neither helical domain alone did. However, transcriptional activation per se was not sufficient because a second activation domain of AF10 was neither required nor competent for transformation. The requirement for alpha-helical transcriptional effector domains is similar to the oncogenic contributions of unrelated MLL partners ENL and ELL, suggesting a general mechanism of myeloid leukemogenesis by a subset of MLL fusion proteins, possibly through specific recruitment of the transcriptional machinery.

Amino Acid Motifs↗

PubMed2002

The MLL fusion partner AF10 binds GAS41, a protein that interacts with the human SWI/SNF complex.

The AF10 gene encodes a putative transcription factor containing an N-terminal LAP/PHD zinc finger motif, a functional nuclear localization signal, an AT-hook domain, and a leucine zipper toward the C-terminus. AF10 is involved in 2 distinct chromosomal translocations associated with hematologic malignancy. The chimeric fusion proteins MLL/AF10 and CALM/AF10, resulting from the t(10;11)(p12;q23) and the t(10;11)(p12;q14), respectively, consistently retain the leucine zipper motif of AF10. This part of the C-terminal region was used as bait in a yeast 2 hybrid screening of a testis complementary DNA library. The leucine zipper interacted with GAS41, a protein previously identified as the product of an amplified gene in a glioblastoma. GAS41 shows significant homology to the Saccharomyces cerevisiae protein ANC1 and to the human MLL fusion partners AF9 and ENL. The interaction was confirmed in vivo. Furthermore, the study showed by coimmunoprecipitation that GAS41 interacts with INI1 (Integrase Interactor 1) and that INI1 was present in the AF10 immunoprecipitate. INI1 is the human homologue of the yeast SNF5 protein, a component of the SWI/SNF complex, which acts to remodel chromatin and to modulate transcription. The retention of the leucine zipper in the MLL and CALM fusions suggests that a key feature of these chimeric proteins may be their ability to interfere in normal gene regulation through interaction with the adenosine triphosphate-dependent chromatinremodeling complexes.

Amino Acid Sequence↗

PubMed2002

Application of multiplex fluorescence in situ hybridization in the cytogenetic analysis of primary gastric carcinoma.

The different genetic alterations observed in diffuse and intestinal types of gastric cancer suggest that these two pathological types may represent different disease entities. We present two cases of primary gastric carcinoma, a well-differentiated intestinal type adenocarcinoma and a poorly differentiated diffuse type adenocarcinoma, both studied by a 24-color multiplex fluorescence in situ hybridization technique (M-FISH). The well-differentiated intestinal type adenocarcinoma exhibited fewer structural abnormalities with five noncomplex translocations, deletions of chromosomes 5q, 6q, and 17q and an i(8q). In the case of poorly differentiated diffuse carcinoma, structural abnormalities predominated and normal homologues were mostly absent. But there were also similarities between the two cases: translocations on 1p and 9p; structural abnormalities of chromosome 8 with consistent loss of 8p; structural abnormalities of 12q; partial loss of chromosome 17 and 18; and polysomy of chromosome 20. This study shows that M-FISH is valuable in identifying hidden structural abnormalities and could, therefore, be useful in the investigation of primary solid tumors.

Adenocarcinoma↗

PubMed2002

Novel chromosome findings in bladder cancer cell lines detected with multiplex fluorescence in situ hybridization.

Bladder cancer is a common neoplasm worldwide, consisting mainly of transitional cell carcinomas, while squamous, adenocarcinoma, and sarcomatoid bladder cancers account for the remaining cases. In the present study, multiplex fluorescence in situ hybridization (M-FISH) has been used to characterize chromosome rearrangements in eight transitional and one squamous cell carcinoma cell line, RT112, of UMUC-3, 5637, CAT(wil), FGEN, EJ28, J82, 253J, and SCaBER. Alterations of chromosome 9 are the most frequent cytogenetic and molecular findings in transitional cell carcinomas of all grades and stages, while changes of chromosomes 3, 4, 8, 9, 11, 14, and 17 are also frequently observed. In the present study, alterations previously described, including del(8)(p10), del(9)(p10), del(17)(p10), and overrepresentation of chromosome 20, as well as several novel findings, were observed. These novel findings were a del(15)(q15) and isochromosome 14q, both occurring in three of nine cell lines examined. These abnormalities may reflect changes in bladder tumor biology. M-FISH represents an effective preliminary screening tool for the characterization of complex tumor karyotypes.

Carcinoma, Squamous Cell↗

PubMed2002

Comparative genomic hybridization and multiplex-fluorescence in situ hybridization: an appraisal in elderly patients with acute myelogenous leukemia.

Comparative genomic hybridization (CGH) and multiplex-fluorescence in situ hybridization (M-FISH) were used to evaluate the presentation karyotype in 15 and 18 patients respectively, aged >/=60 years, with acute myeloid leukemia (AML). Conventional G-banded analysis was performed in all patients prior to evaluation. Comparative genomic hybridization confirmed the G-banded karyotype fully in 12 patients and partially in two patients. Normal CGH profiles were observed in patients with a normal karyotype and in one patient with a balanced chromosomal translocation as the sole cytogenetic aberration. Multiplex-fluorescence in situ hybridization provided additional information in two patients with a complex karyotype, but failed to detect a telomeric translocation in one patient. Eight patients with normal G-banded karyotypes appeared normal by M-FISH. These results demonstrate that both CGH and M-FISH analysis correlate well with the G-banded karyotype in AML. Furthermore, although additive cytogenetic data was not provided by either technique in cases with normal karyotype, DNA copy number change and cryptic translocations below the resolution of CGH and M-FISH may still be the initiating event for leukemogenesis for these patients.

Aged↗

PubMed2002

JH probe real-time quantitative polymerase chain reaction assay for Bcl-2/IgH rearrangements.

Follicular lymphoma (FL) characteristically bears the t(14;18)(q32;q21). However, only approximately 75% of the consequent Bcl-2 breakpoints lie within the major breakpoint region (MBR) or the minor cluster region (mcr). While these can be quantified by cluster region-specific real-time quantitative polymerase chain reaction (RQ-PCR), a significant proportion of cases are left requiring a customized approach. Therefore, an RQ-PCR assay for the quantification of Bcl-2/IgH breakpoints has been developed that uses germline JH TaqMan probes and germline JH primers in combination with customized forward primers. Validation of this approach by comparison with an established MBR RQ-PCR showed both techniques to be concordant across a wide range of copy numbers with a sensitivity of five copies per 10(5) cells. In addition, to generate standard curves equating to diverse Bcl-2/IgH rearrangements, a strategy for using placental DNA as a surrogate standard was devised. The performance of the assay in detecting molecular evidence of disease in sequential biopsies from five patients (three with atypical Bcl-2/IgH breakpoints identified by long-range or inverse PCR, one MBR+ and one mcr+) was tested. This alternative approach represents a sensitive and specific means of quantifying common and atypical Bcl-2/IgH rearrangements and maximizes the number of patients with FL suitable for molecular monitoring.

Chromosome Breakage↗